Intracellular calcium is a critical signaling molecule in the brain for many functions including neuronal development and synaptic plasticity. A family of Ca2+/calmodulin-regulated protein kinases (CaMKs) are key regulators of several of these events. During the past several years we identified regulatory functions of CaM-KK and its downstream effector CaMKI in the morphology and motility of axonal growth cones and basal axonal outgrowth, activity-dependent dendritic arborization and early-phase long-term potentiation. In the current application we will focus on two critical aspects of neuronal development, axon specification and formation of dendritic spines, for which we have preliminary evidence for involvement of CaMKK and CaMKI. We will analyze the activation state of CaMKK and CaMKI isoforms in different subcellular compartments of cultured hippocampal neurons using several approaches including FRET analysis. From proteomic analysis, we have evidence for the existence of a CaMKK signalsome containing beta-PIX and GIT1, and FIX is an in vitro substrate for CaMKI. The role of this signaling complex, as well as several other CaMKI substrates (Numb and stathmin 2) known to regulate the actin and microtubule cytoskelton, in axon specification and spinogenesis will be explored. Phosphorylation of these proteins by CaMKI in neurons will be studied using phospho-specific antibodies. These signaling pathways from CaMKI to axonogensis and spinogenesis will be defined using multiple, independent approaches including dominant-interferring and constitutively-active constructs, pharmacological inhibitors, and RNA interference. The primary experimental system will be cultured hippocampal neurons, but results will be extended into cultured hippocampal slices where possible. Identifying the fundamental signaling pathwaysthat modulate these essential neuronal functions is foundational to understanding normal and pathological brain functions including axon regeneration and mental retardation. Structural abnormalities in dendritic arborization and spine formation, both in human patients and in mouse models, are common to numberous forms of mental retardation including Fragile X, fetal alcohol, Down's and Rett syndromes as well as several evironmental causes of retardation. Thus, these studies have strong implications for underlying causes of mental diseases.